The present invention relates to novel members of the Tumor Necrosis Factor (TNF) receptor family. More specifically, isolated nucleic acid molecules are provided encoding a human TNF receptor-related protein, referred to herein as the TR11 receptor of FIGS. 1A and 1B, and two splice variants thereof referred to herein as the TR11SV1 and TR11SV2 receptors, of FIGS. 2A and 2B and 3A and 3B, respectively, each having considerable homology to murine glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR). TR11, TR11SV1, and TR11SV2 polypeptides are also provided. Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of the activities of TR11, TR11SV1, and TR11SV2 receptor polypeptides and diagnostic methods for detecting TR11 receptor gene expression.
Human tumor necrosis factors alpha (TNF-alpha) and beta (TNF-beta or lymphotoxin) are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol., 7:625-655 (1989)).
Tumor necrosis factor (TNF-alpha and TNF-beta) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic, cytokine playing important roles in a host of biological processes and pathologies. To date, there are ten known members of the TNF-related cytokine family, TNF-alpha, TNF-beta (lymphotoxin-alpha), LT-beta, TRAIL and ligands for the Fas receptor, CD30, CD27, CD40 (also known as CDw40), OX40 and 4-1BB receptors. These proteins have conserved C-terminal sequences and variable N-terminal sequences which are often used as membrane anchors, with the exception of TNF-beta. Both TNF-alpha and TNF-beta function as homotrimers when they bind to TNF receptors.
TNF is produced by a number of cell types, including monocytes, fibroblasts, T-cells, natural killer (NK) cells and predominately by activated macrophages. TNF-alpha has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata, et al., J. Immunol. 136:2483 (1987)), growth regulation, vascular endothelium effects and metabolic effects. TNF-alpha also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation. In addition, TNF-alpha up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-alpha and the Fas ligand have also been shown to induce programmed cell death.
TNF-beta has many activities, including induction of an antiviral state and tumor necrosis, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle, N. and Homer, R., Prog. Allergy 40:162-182 (1988)).
Both TNF-alpha and TNF-beta are involved in growth regulation and interact with hemopoietic cells at several stages of differentiation, inhibiting proliferation of various types of precursor cells, and inducing proliferation of immature myelomonocytic cells (Porter, A., Tibtech 9:158-162 (1991)).
Recent studies with xe2x80x9cknockoutxe2x80x9d mice have shown that mice deficient in TNF-beta production show abnormal development of the peripheral lymphoid organs and morphological changes in spleen architecture (reviewed by Aggarwal, et al., Eur Cytokine Netw, 7:93-124 (1996)). With respect to the lymphoid organs, the popliteal, inguinal, para-aortic, mesenteric, axillary and cervical lymph nodes failed to develop in TNF-betaxe2x88x92/xe2x88x92mice. In addition, peripheral blood from TNF-betaxe2x88x92/xe2x88x92mice contained a three fold reduction in white blood cells as compared to normal mice. Peripheral blood from TNF-betaxe2x88x92/xe2x88x92mice, however, contained four fold more B cells as compared to their normal counterparts. Further, TNF-beta, in contrast to TNF-alpha, has been shown to induce proliferation of EBV-infected B cells. These results indicate that TNF-beta is involved in lymphocyte development.
The first step in the induction of the various cellular responses mediated by TNF-alpha or TNF-beta is their binding to specific cell surface or soluble receptors. Two distinct TNF receptors of approximately 55-KDa (TNF-RI) and 75-KDa (TNF-RII) have been identified (Hohman, et al., J. Biol. Chem., 264:14927-14934 (1989)), and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher, et al., Cell, 61:351 (1990)). Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions.
These molecules exist not only in cell bound forms, but also in soluble forms, consisting of the cleaved extra-cellular domains of the intact receptors (Nophar, et al., EMBO Journal, 9:3269-76 (1990)) and otherwise intact receptors wherein the transmembrane domain is lacking. The extracellular domains of TNF-RI and TNF-RII share 28% identity and are characterized by four repeated cysteine-rich motifs with significant intersubunit sequence homology. The majority of cell types and tissues appear to express both TNF receptors and both receptors are active in signal transduction, however, they are able to mediate distinct cellular responses. Further, TNF-RII was shown to exclusively mediate human T-cell proliferation by TNF as shown in PCT WO 94/09137.
TNF-RI dependent responses include accumulation of C-FOS, IL-6, and manganese superoxide dismutase mRNA, prostaglandin E2 synthesis, IL-2 receptor and MHC class I and II cell surface antigen expression, growth inhibition, and cytotoxicity. TNF-RI also triggers second messenger systems such as phospholipase A, protein kinase C, phosphatidylcholine-specific phospholipase C and sphingomyelinase (Pfefferk, et al., Cell, 73:457-467 (1993)).
Several interferons and other agents have been shown to regulate the expression of TNF receptors. Retinoic acid, for example, has been shown to induce the production of TNF receptors in some cells type while down regulating production in other cells. In addition, TNF-alpha has been shown to affect the localization of both types of receptor. TNF-alpha induces internalization of TNF-RI and secretion of TNF-RII (reviewed in Aggarwal, et al., supra). Thus, the production and localization of both TNF-Rs are regulated by a variety of agents.
Both the yeast two hybrid system and co-precipitation and purification have been used to identify ligands which associate with both types of the TNF-Rs (reviewed by Aggarwal, et al., supra; Vandenabeele, et al., Trends in Cell Biol. 5:392-399 (1995)). Several proteins have been identified which interact with the cytoplasmic domain of a murine TNF-R. Two of these proteins appear to be related to the baculovirus inhibitor of apoptosis, suggesting a direct role for TNF-R in the regulation of programmed cell death.
Thus, there is a need for polypeptides that function as receptors for cytokines and cytokine-like molecules which are involved in the regulation of cellular processes such as cell-growth and differentiation, since disturbances of such regulation may be involved in disorders relating to hemostasis, angiogenesis, tumor metastisis, cellular migration, and neurogenesis. Therefore, there is a need for identification and characterization of such human polypeptides which can play a role in detecting, preventing, ameliorating, regulating or correcting such disorders.
The present invention provides isolated nucleic acid molecules comprising or alternatively consisting of, polynucleotides encoding TR11, TR11SV1, and TR11SV2 receptors having the amino acid sequences shown in FIGS. 1A and 1B (SEQ ID NO:2), 2A and 2B (SEQ ID NO:4), and 3A and 3B (SEQ ID NO:6), respectively, or the amino acid sequences encoded by the cDNA clones encoding the TR11, TR11SV1, and TR11SV2 receptors, respectively, deposited as ATCC Deposit Numbers 209341, 209343 and 209342, respectively, on Oct. 7, 1997. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TR11, TR11SV1, and TR11SV2 polypeptides or peptides by recombinant techniques.
The invention further provides isolated TR11, TR11SV1, and TR11SV2 polypeptides having amino acid sequences encoded by the polynucleotides described herein.
In certain embodiments, TR11, TR11SV1, and/or TR11SV2 polypeptides of the invention, or agonists thereof, are administered, to treat, prevent, prognose and/or diagnose an immunodeficiency (e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), DiGeorge anomaly, Bruton""s disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CV1D) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaclobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency) or conditions associated with an immunodeficiency.
In a specific embodiment, TR11, TR11SV1, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose common variable immunodeficiency.
In a specific embodiment, TR11, TR11SV1, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose X-linked agammaglobulinemia.
In another specific embodiment, TR11, TR11SV1, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose severe combined immunodeficiency (SCID).
In another specific embodiment, TR11, TR11SV1, and/or TR11SV2 polypeptides or polynucleotides of the invention, or, agonists thereof, is administered to treat, prevent, prognose and/or diagnose Wiskott-Aldrich syndrome.
In another specific embodiment, TR11, TR11SV1, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.
In another embodiment, TR11, TR11SV1, and/or TR11SV2 antagonists (e.g., an anti-TR11, TR11SV1, and/or TR11SV2 antibody), are administered to treat, prevent, prognose and/or diagnose an autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter""s Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto""s thyroiditis, Goodpasture""s syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves"" Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison""s disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren""s syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulamatous, degenerative, and atrophic disorders) or conditions associated with an autoimmune disease. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, prognosed and/or diagnosed using anti-TR11, TR11SV1, and/or TR11SV2 antibodies and/or other antagonist of the invention. In another specific preferred embodiment, systemic lupus erythemosus is treated, prevented, prognosed, and/or diagnosed using anti-TR11, TR11SV1, and/or TR11SV2 antibodies and/or other antagonist of the invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, prognosed, and/or diagnosed using anti-TR11, TR11SV1, and/or TR11SV2 antibodies and/or other antagonist of the invention. In another specific preferred embodiment IgA nephropathy is treated, prevented, prognosed and/or diagnosed using anti-TR11, TR11SV1, and/or TR11SV2 antibodies and/or other antagonist of the invention. In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, prognosed and/or diagnosed using anti-TR11, TR11SV1, and/or TR11 SV2 antibodies.
The invention further provides compositions comprising a TR11, TR11SV1, and/or TR11SV2 polynucleotide, a TR11, TR11SV1, and/or TR11SV2 polypeptide, and/or an anti-TR11, TR11SV1, and/or TR11SV2 antibody, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism. In preferred embodiments, the compositions of the invention comprise a TR11, TR11SV1, and/or TR11SV2 polynucleotide for expression of a TR11, TR11SV1, and/or TR11SV2 polypeptide in a host organism for treatment of disease. In a most preferred embodiment, the compositions of the invention comprise a TR11, TR11SV1, and/or TR11SV2 polynucleotide for expression of a TR11, TR11SV1, and/or TR11SV2 polypeptide in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of a TR11, TR11SV1, and/or TR11SV2 gene (e.g., expression to enhance the normal B-cell function by expanding B-cell numbers or increasing B cell lifespan; or expression to enhance the normal T cell function by expanding T cell numbers or increasing T cell lifespan).
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by TR11, TR11SV1, and TR11SV2 receptors, which involves contacting cells which express TR11, TR11SV1 or TR11SV2 receptors with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on the binding of cellular ligands to TR11, TR11SV1, and TR11SV2 receptors. In particular, the method involves contacting TR11, TR11SV1, and TR11SV2 receptors with a ligand polypeptide and a candidate compound and determining whether ligand binding to the TR11, TR11SV1, and TR11SV2 receptors is increased or decreased due to the presence of the candidate compound.
The invention further provides a diagnostic method useful during diagnosis or prognosis of one or more disease states resulting from aberrant cell proliferation due to alterations in TR11, TR11SV1, and TR11SV2 receptor expression.
An additional aspect of the invention is related to a method for treating, detecting, and/or preventing an individual in need of an increased level of a TR11, TR11SV1 or TR11SV2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of isolated. TR11, TR11SV1 or TR11SV2 polypeptides of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating, detecting, and/or preventing an individual in need of a decreased level of a TR11, TR11SV1 or TR11SV2 receptor activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a TR11, TR11 SV1 or TR11SV2 receptor antagonist.
The invention additionally provides soluble forms of the polypeptides of the present invention. Soluble peptides are defined by amino acid sequences wherein the sequence comprises the polypeptide sequence lacking a transmembrane domain. Such soluble forms of the TR11, TR11SV1, and TR11SV2 receptors are useful as antagonists of the membrane bound forms of the receptors.